Treatment of hidradenitis suppurativa using jak inhibitors

ABSTRACT

The present application provides methods of treating hidradenitis suppurativa in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound which inhibits JAK1 and/or JAK2, or a pharmaceutically acceptable salt thereof.

The present application claims the benefit of U.S. ProvisionalApplication No. 62/650,600, filed Mar. 30, 2018, which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present application provides methods for the treatment hidradenitissuppurativa (HS) using compounds that modulate the activity of Januskinase (JAK) 1 and/or 2.

BACKGROUND

Protein kinases (PKs) regulate diverse biological processes includingcell growth, survival, differentiation, organ formation, morphogenesis,neovascularization, tissue repair, and regeneration, among others.Protein kinases also play specialized roles in a host of human diseasesincluding cancer. Cytokines, low-molecular weight polypeptides orglycoproteins, regulate many pathways involved in the host inflammatoryresponse to sepsis. Cytokines influence cell differentiation,proliferation and activation, and can modulate both pro-inflammatory andanti-inflammatory responses to allow the host to react appropriately topathogens. Signaling of a wide range of cytokines involves the Januskinase family (JAKs) of protein tyrosine kinases and Signal Transducersand Activators of Transcription (STATs). There are four known mammalianJAKs: JAK1 (Janus kinase-1), JAK2, JAK3 (also known as Janus kinase,leukocyte; JAKL; and L-JAK), and TYK2 (protein-tyrosine kinase 2).

Cytokine-stimulated immune and inflammatory responses contribute topathogenesis of diseases: pathologies such as severe combinedimmunodeficiency (SCID) arise from suppression of the immune system,while a hyperactive or inappropriate immune/inflammatory responsecontributes to the pathology of autoimmune diseases (e.g., asthma,systemic lupus erythematosus, thyroiditis, myocarditis), and illnessessuch as scleroderma and osteoarthritis (Ortmann, R. A., T. Cheng, et al.(2000) Arthritis Res 2(1): 16-32).

Deficiencies in expression of JAKs are associated with many diseasestates. For example, Jak1−/− mice are runted at birth, fail to nurse,and die perinatally (Rodig, S. J., M. A. Meraz, et al. (1998) Cell93(3): 373-83). Jak2−/− mouse embryos are anemic and die around day 12.5postcoitum due to the absence of definitive erythropoiesis.

The JAK/STAT pathway, and in particular all four JAKs, are believed toplay a role in the pathogenesis of asthmatic response, chronicobstructive pulmonary disease, bronchitis, and other relatedinflammatory diseases of the lower respiratory tract. Multiple cytokinesthat signal through JAKs have been linked to inflammatorydiseases/conditions of the upper respiratory tract, such as thoseaffecting the nose and sinuses (e.g., rhinitis and sinusitis) whetherclassically allergic reactions or not. The JAK/STAT pathway has alsobeen implicated in inflammatory diseases/conditions of the eye andchronic allergic responses.

Activation of JAK/STAT in cancers may occur by cytokine stimulation(e.g. IL-6 or GM-CSF) or by a reduction in the endogenous suppressors ofJAK signaling such as SOCS (suppressor or cytokine signaling) or PIAS(protein inhibitor of activated STAT) (Boudny, V., and Kovarik, J.,Neoplasm. 49:349-355, 2002). Activation of STAT signaling, as well asother pathways downstream of JAKs (e.g., Akt), has been correlated withpoor prognosis in many cancer types (Bowman, T., et al. Oncogene19:2474-2488, 2000). Elevated levels of circulating cytokines thatsignal through JAK/STAT play a causal role in cachexia and/or chronicfatigue. As such, JAK inhibition may be beneficial to cancer patientsfor reasons that extend beyond potential anti-tumor activity. JAK2tyrosine kinase can be beneficial for patients with myeloproliferativedisorders, e.g., polycythemia vera (PV), essential thrombocythemia (ET),myeloid metaplasia with myelofibrosis (MMM) (Levin, et al., Cancer Cell,vol. 7, 2005: 387-397). Inhibition of the JAK2V617F kinase decreasesproliferation of hematopoietic cells, suggesting JAK2 as a potentialtarget for pharmacologic inhibition in patients with PV, ET, and MMM.

Inhibition of the JAKs may benefit patients suffering from skin immunedisorders such as psoriasis, and skin sensitization. The maintenance ofpsoriasis is believed to depend on a number of inflammatory cytokines inaddition to various chemokines and growth factors (JCI, 113:1664-1675),many of which signal through JAKs (Adv Pharmacol. 2000; 47:113-74).

Thus, new or improved agents which inhibit kinases such as JAKs arecontinually needed for developing new and more effective pharmaceuticalsthat are aimed at augmentation or suppression of the immune andinflammatory pathways, such as the treatment of hidradenitissuppurativa. This application is directed to that need and others.

SUMMARY

The present application provides methods of treating hidradenitissuppurativa in a patient in need thereof, comprising administering tothe patient a therapeutically effective amount of a compound whichinhibits JAK1 and/or JAK2, or a pharmaceutically acceptable saltthereof.

In some embodiments, the compound or salt is selective for JAK1 andJAK2, which is selective over JAK3 and TYK2.

In some embodiments, the compound or salt is selective for JAK1 overJAK2, JAK3, and TYK2.

In some embodiments, the compound is ruxolitinib, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the compound is ruxolitinib, or a pharmaceuticallyacceptable salt thereof, wherein one or more hydrogen atoms are replacedby deuterium atoms.

In some embodiments, the salt is ruxolitinib phosphate.

In some embodiments, the compound is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,or a pharmaceutically acceptable salt thereof.

In some embodiments, the salt is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileadipic acid salt.

In some embodiments, the compound is4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide,or a pharmaceutically acceptable salt thereof.

In some embodiments, the salt is4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamidephosphoric acid salt.

In some embodiments, the compound or salt is administered at a dosage of15, 30, 60 or 90 mg on a free base basis.

In some embodiments, the compound is((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile,or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrilemonohydrate.

In some embodiments, the methods further comprise administering anadditional therapeutic agent (e.g., an antibiotic, a retinoid, acorticosteroid, an anti-TNF-alpha agent, or an immunosuppressant).

In some embodiments, the administrating of the compound or salt istopical. In some embodiments, the administering of the compound or saltis oral.

In some embodiments, the method results in a 10%, 20%, 30%, 40%, or 50%improvement in HiSCR (Hidradenitis Suppurativa Clinical Response).

The present application also provides a compound which inhibits JAK1and/or JAK2, or a pharmaceutically acceptable salt thereof, for use intreating hidradenitis suppurativa.

The present application further provides use of a compound whichinhibits JAK1 and/or JAK2, or a pharmaceutically acceptable saltthereof, for preparation of a medicament for use in treatment ofhidradenitis suppurativa.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the individual gene expression values (MFI) for JAK1,for each experimental replicate in keratinocytes simulated with TNFα andIFN-γ in the presence/absence of Compounds A-D. Keratinocytes werestimulated with TNFα (25 ng/mL) and IFNγ (25 ng/mL) in thepresence/absence of increasing concentrations of JAK inhibitors. Dataare presented as JAK1 expression levels for each group.

FIG. 2 illustrates the individual gene expression values (MFI) for JAK2,for each experimental replicate in keratinocytes simulated with TNFα andIFN-γ in the presence/absence of Compounds A-D. Keratinocytes werestimulated with TNFα (25 ng/mL) and IFNγ (25 ng/mL) in thepresence/absence of increasing concentrations of JAK inhibitors. Dataare presented as JAK2 expression levels for each group.

FIG. 3 illustrates the individual gene expression values (MFI) for IL-1αfor each experimental replicate in keratinocytes simulated with TNFα andIFN-γ in the presence/absence of Compounds A-D. Keratinocytes werestimulated with TNFα (25 ng/mL) and IFNγ (25 ng/mL) in thepresence/absence of increasing concentrations of JAK inhibitors. Dataare presented as IL-1α expression levels for each group.

FIG. 4 illustrates the individual gene expression values (MFI) for IL-6,for each experimental replicate in keratinocytes simulated with TNFα andIFN-γ in the presence/absence of Compounds A-D. Keratinocytes werestimulated with TNFα (25 ng/mL) and IFNγ (25 ng/mL) in thepresence/absence of increasing concentrations of JAK inhibitors. Dataare presented as IL-6 expression levels for each group.

FIG. 5 illustrates the individual protein concentrations (pg/mL) forIL-1α, for each experimental replicate in keratinocytes simulated withTNFα and IFN-γ in the presence/absence of Compounds A-D. Keratinocyteswere stimulated with TNFα (25 ng/mL) and IFNγ (25 ng/mL) in thepresence/absence of increasing concentrations of JAK inhibitors. Dataare presented as IL-1α concentrations for each group.

FIG. 6 illustrates the individual protein concentrations (pg/mL) forIL-6, for each experimental replicate in keratinocytes simulated withTNFα and IFN-γ in the presence/absence of Compounds A-D. Keratinocyteswere stimulated with TNFα (25 ng/mL) and IFNγ (25 ng/mL) in thepresence/absence of increasing concentrations of JAK inhibitors. Dataare presented as IL-6 concentrations for each group.

FIG. 7 illustrates the gene expression (MFI) of JAK1, JAK3, and TYK2 inthe skin of healthy controls and subjects with hidradenitis suppurativa.Data are presented as JAK1, JAK3, or TYK2 gene expression levels foreach Healthy Control (n=4) and Hidradenitis Suppurativa (n=41) subject.

FIG. 8 illustrates the gene expression (MFI) of STAT1, STAT2, and STAT3in the skin of healthy controls and subjects with hidradenitissuppurativa. Data are presented as STAT1, STAT2, or STAT3 geneexpression levels for each Healthy Control (n=4) and HidradenitisSuppurativa (n=41) subject.

FIG. 9 illustrates the gene expression (MFI) of IRAK1, IRAK2, and IRAK4in the skin of healthy controls and subjects with hidradenitissuppurativa. Data are presented as IRAK1, IRAK2, or IRAK4 geneexpression levels for each Healthy Control (n=4) and HidradenitisSuppurativa (n=41) subject.

DETAILED DESCRIPTION

The present application provides, inter alia, a method of treatinghidradenitis suppurativa in a patient in need thereof, comprisingadministering a therapeutically effective amount of compound whichinhibits JAK1 and/or JAK2, or a pharmaceutically acceptable saltthereof.

The method described herein utilize compound or salts that areinhibitors of JAK1 and/or JAK2. In some embodiments, the compound is:

-   ruxolitinib;-   ruxolitinib, wherein one or more hydrogen atoms are replaced by    deuterium atoms;-   {1-{1-[3-Fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl-N}-[4-fluoro-2-(trifluoromethyl)phenyl]piperidine-1-carboxamide;-   [3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-1-(1-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)azetidin-3-yl]acetonitrile;-   4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,    1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide;-   ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile;-   3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile;-   3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile;-   4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile;-   4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile;-   [trans-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-3-(4-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperazin-1-yl)cyclobutyl]acetonitrile;-   {trans-3-(4-{[4-[(3-hydroxyazetidin-1-yl)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   {trans-3-(4-{[4-{[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   {trans-3-(4-{[4-{[(2R)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile;-   5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide;-   4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide;-   5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide;-   {1-(cis-4-{[6-(2-hydroxyethyl)-2-(trifluoromethyl)pyrimidin-4-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   {1-(cis-4-{[4-[(ethylamino)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   {1-(cis-4-{[4-(1-hydroxy-1-methylethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   {1-(cis-4-{[4-{[(3R)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   {1-(cis-4-{[4-{[(3S)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;-   {trans-3-(4-{[4-({[(1S)-2-hydroxy-1-methylethyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   {trans-3-(4-{[4-({[(2R)-2-hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile    {trans-3-(4-{[4-({[(2S)-2-hydrox;    ypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   {trans-3-(4-{[4-(2-hydroxyethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;-   or a pharmaceutically acceptable salt of any of the aforementioned.

In some embodiments, the compound or salt is selective for JAK1 and JAK2over JAK3 and TYK2. In some embodiments, the compound is3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,or a pharmaceutically acceptable salt thereof. In some embodiments, thecompound is(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile(ruxolitinib), or a pharmaceutically acceptable salt thereof.Ruxolitinib has an IC₅₀ of less than 10 nM at 1 mM ATP (assay A) at JAK1and JAK2.3-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand ruxolitinib can be made by the procedure described in U.S. Pat. No.7,598,257 (Example 67), filed Dec. 12, 2006, which is incorporatedherein by reference in its entirety. In some embodiments, the inhibitorof JAK1 and/or JAK2 is(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrilephosphoric acid salt. The phosphoric acid salt can be made as describedin U.S. Pat. No. 8,722,693, which is incorporated herein by reference inits entirety.

In some embodiments, the compound or salt is a JAK1 inhibitor. In someembodiments, the compound or salt is selective for JAK1 over JAK2, JAK3and TYK2. For example, some of the compounds described herein, or apharmaceutically acceptable salt thereof, preferentially inhibit JAK1over one or more of JAK2, JAK3, and TYK2. JAK1 plays a central role in anumber of cytokine and growth factor signaling pathways that, whendysregulated, can result in or contribute to disease states. Forexample, IL-6 levels are elevated in rheumatoid arthritis, a disease inwhich it has been suggested to have detrimental effects (Fonesca, etal., Autoimmunity Reviews, 8:538-42, 2009). Because L-6 signals, atleast in part, through JAK1, IL-6 can be indirectly through JAK1inhibition, resulting in potential clinical benefit (Guschin, et al.Embo J 14:1421, 1995; Smolen, et al. Lancet 371:987, 2008). Moreover, insome cancers JAK1 is mutated resulting in constitutive undesirable tumorcell growth and survival (Mullighan, Proc Natl Acad Sci USA. 106:9414-8,2009; Flex, J Exp Med. 205:751-8, 2008). In other autoimmune diseasesand cancers, elevated systemic levels of inflammatory cytokines thatactivate JAK1 may also contribute to the disease and/or associatedsymptoms. Therefore, patients with such diseases may benefit from JAK1inhibition. Selective inhibitors of JAK1 may be efficacious whileavoiding unnecessary and potentially undesirable effects of inhibitingother JAK kinases.

Hidradenitis suppurativa is characterized by significant skininflammation; however, there are limited publications outlining theinflammation (Hoffman et al., PLOS One, Sep. 28, 2018,https://doi.org/10.1371/journal.pone.0203672). Presented herein areExamples that support the hypothesis that the inflammation is driven, inlarge part, by JAK/STAT mediated pathways. Examples C, D and Eillustrate elevated levels of JAK/STAT gene expression in the skin of HSpatients compared to healthy skin. Further, Examples C, D and E showthat pro-inflammatory cytokines which are known to be elevated in HS(TNF-alpha and IFN-gamma) induce the JAK/STAT pathway in culturedkeratinocytes and that this induction can be reduced by the addition ofJAK inhibitors. Therefore, patients with HS may benefit from JAK1inhibition. Selective inhibitors of JAK1 may be efficacious whileavoiding unnecessary and potentially undesirable effects of inhibitingother JAK kinases.

In some embodiments, the compound or salt inhibits JAK1 preferentiallyover JAK2 (e.g., have a JAK2/JAK1 IC₅₀ ratio>1). In some embodiments,the compounds or salts are about 10-fold more selective for JAK1 overJAK2. In some embodiments, the compounds or salts are about 3-fold,about 5-fold, about 10-fold, about 15-fold, or about 20-fold moreselective for JAK1 over JAK2 as calculated by measuring IC₅₀ at 1 mM ATP(see Example A).

In some embodiments, the JAK1 inhibitor is a compound of Table 1, or apharmaceutically acceptable salt thereof. The compounds in Table 1 areselective JAK1 inhibitors (selective over JAK2, JAK3, and TYK2). TheIC₅₀ values obtained by the method of Example A at 1 mM ATP are shown inTable 1.

TABLE 1 JAK1 Comp. IC₅₀ JAK2/ No. Prep. Name Structure (nM) JAK1  1 US2011/ 0224190 (Example 1) {1-{1-[3-Fluoro-2- (trifluoromethyl)iso-nicotinoyl]piperidin-4- yl}-3- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10  2 US 2011/ 0224190 (Example 154) 4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-1-yl}-N-[4-fluoro-2- (trifluoromethyl)phenyl] piperidine-1- carboxamide

+ >10  3 US 2011/ 0224190 (Example 85) [3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]-1-(1-{[2- (trifluoro-methyl)pyrimidin-4- yl]carbonyl}piperidin-4- yl)azetidin-3-yl]acetonitrile

+ >10  4 US 2014/ 0343030 (Example 7) 4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H- 4,4′-bipyrazol-1- yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2- trifluoro-1- methylethyl]benzamide

+++ >10  5 US 2014/ 0121198 (Example 20) ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H- imidazo[4,5- d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H- pyran-2-yl)acetonitrile

++ >10  6 US 2010/ 0298334 (Example 2)^(a) 3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4- (7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]propanenitrile

+ >10  7 US 2010/ 0298334 (Example 13c) 3-(1-[1,3]oxazolo[5,4-b]pyridin-2- ylpyrrolidin-3-yl)-3-[4- (7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1- yl]propanenitrile

+ >10  8 US 2011/ 0059951 (Example 12) 4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1- yl]propyl}piperazin-1-yl)carbonyl]-3- fluorobenzonitrile

+ >10  9 US 2011/ 0059951 (Example 13) 4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrrol-1- yl]propyl}piperazin-1-yl)carbonyl]-3- fluorobenzonitrile

+ >10 10 US 2012/ 0149681 (Example 7b) [trans-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]-3-(4-{[2- (trifluoromethyl)pyrimidin-4- yl]carbonyl}piperazin- 1-yl)cyclo- butyl]acetonitrile

+ >10 11 US 2012/ 0149681 (Example 157) {trans-3-(4-{[4-[(3-hydroxyazetidin-1- yl)methyl]-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclo- butyl}acetonitrile

+ >10 12 US 2012/ 0149681 (Example 161) {trans-3-(4-{[4-{[(2S)-2-(hydroxy- methyl)pyrrolidin- 1-yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 13 US 2012/ 0149681 (Example 162) {trans-3-(4-{[4-{[(2R)-2-(hydroxy- methyl)pyrrolidin- 1-yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 14 US 2012/ 0149682 (Example 20)^(b) 4-(4-{3-[(dimethyl-amino)methyl]-5- fluorophenoxy}piperidin- 1-yl)-3-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1- yl]butanenitrile

+ >10 15 US 2013/ 0018034 (Example 18) 5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-1-yl}-N- isopropylpyrazine-2- carboxamide

+ >10 16 US 2013/ 0018034 (Example 28) 4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N- [(1S)-2,2,2-trifluoro-1- methylethyl]benzamide

+ >10 17 US 2013/ 0018034 (Example 34) 5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3- b]pyridin-4-yl)-1H- pyrazol-1-yl]azetidin-1- yl}-N-isopropylpyrazine-2- carboxamide

+ >10 18 US 2013/ 0045963 (Example 45) {1-(cis-4-{[6-(2-hydroxyethyl)-2- (trifluoromethyl) pyrimidin-4- yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-3-yl}acetonitrile

+ >10 19 US 2013/ 0045963 (Example 65) {1-(cis-4-{[4-[(ethylamino)methyl]-6- (trifluoromethyl) pyridin-2-yl]oxy} cyclohexyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-3-yl}acetonitrile

+ >10 20 US 2013/ 0045963 (Example 69) {1-(cis-4-{[4-(1- hydroxy-1-methylethyl)-6- (trifluoromethyl)pyridin- 2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-3-yl}acetonitrile

+ >10 21 US 2013/ 0045963 (Example 95) {1-(cis-4-{[4-{[(3R)-3-hydroxypyrrolidin-1- yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)- 3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10 22 US 2013/ 0045963 (Example 95) {1-(cis-4-{[4-{[(3S)-3-hydroxypyrrolidin-1- yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)- 3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10 23 US 2014/ 0005166 (Example 1) {trans-3-(4-{[4-({[(1S)-2-hydroxy-1-methyl- ethyl]amino}methyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 24 US 2014/ 0005166 (Example 14) {trans-3-(4-{[4-({[(2R)- 2-hydroxypropyl]amino} methyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 25 US 2014/ 0005166 (Example 15) {trans-3-(4-{[4-({[(2S)- 2-hydroxypropyl]amino} methyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H- pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 26 US 2014/ 0005166 (Example 20) {trans-3-(4-{[4-(2-hydroxyethyl)-6- (trifluoromethyl)pyridin- 2-yl]oxy}piperidin-1-yl)-1-[4-(7H- pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]cyclobutyl}acetonitrile

+ >10 + means <10 nM (see Example A for assay conditions) ++ means ≤100nM (see Example A for assay conditions) +++ means ≤300 nM (see Example Afor assay conditions) ^(a)Data for enantiomer 1 ^(b)Data for enantiomer2

In some embodiments, the JAK1 inhibitor is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileadipic acid salt.

The synthesis and preparation of{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileand the adipic acid salt of the same can be found, e.g., in US PatentPubl. No. 2011/0224190, filed Mar. 9, 2011, US Patent Publ. No.2013/0060026, filed Sep. 6, 2012, and US Patent Publ. No. 2014/0256941,filed Mar. 5, 2014, each of which is incorporated herein by reference inits entirety.

In some embodiments, the JAK1 inhibitor is4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide,or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamidephosphoric acid salt.

In some embodiment, the JAK1 is4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamidehydrochloric acid salt.

In some embodiment, the JAK1 is4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamidehydrobromic acid salt.

In some embodiment, the JAK1 is4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamidesulfuric acid salt.

The synthesis and preparation of4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamideand the phosphoric acid salt of the same can be found, e.g., in USPatent Publ. No. US 2014/0343030, filed May 16, 2014, which isincorporated herein by reference in its entirety.

In some embodiments, the JAK1 inhibitor is((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile,or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrilemonohydrate.

Synthesis of((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrileand characterization of the anhydrous and monohydrate forms of the sameare described in US Patent Publ. No. 2014/0121198, filed Oct. 31, 2013and US Patent Publ. No. 2015/0344497, filed Apr. 29, 2015, each of whichis incorporated herein by reference in its entirety.

In some embodiments, the compounds of Table 1 are prepared by thesynthetic procedures described in US Patent Publ. No. 2011/0224190,filed Mar. 9, 2011, US Patent Publ. No. 2014/0343030, filed May 16,2014, US Patent Publ. No. 2014/0121198, filed Oct. 31, 2013, US PatentPubl. No. 2010/0298334, filed May 21, 2010, US Patent Publ. No.2011/0059951, filed Aug. 31, 2010, US Patent Publ. No. 2012/0149681,filed Nov. 18, 2011, US Patent Publ. No. 2012/0149682, filed Nov. 18,2011, US Patent Publ. 2013/0018034, filed Jun. 19, 2012, US Patent Publ.No. 2013/0045963, filed Aug. 17, 2012, and US Patent Publ. No.2014/0005166, filed May 17, 2013, each of which is incorporated hereinby reference in its entirety.

In some embodiments, JAK1 inhibitor is selected from the compounds, orpharmaceutically acceptable salts thereof, of US Patent Publ. No.2011/0224190, filed Mar. 9, 2011, US Patent Publ. No. 2014/0343030,filed May 16, 2014, US Patent Publ. No. 2014/0121198, filed Oct. 31,2013, US Patent Publ. No. 2010/0298334, filed May 21, 2010, US PatentPubl. No. 2011/0059951, filed Aug. 31, 2010, US Patent Publ. No.2012/0149681, filed Nov. 18, 2011, US Patent Publ. No. 2012/0149682,filed Nov. 18, 2011, US Patent Publ. 2013/0018034, filed Jun. 19, 2012,US Patent Publ. No. 2013/0045963, filed Aug. 17, 2012, and US PatentPubl. No. 2014/0005166, filed May 17, 2013, each of which isincorporated herein by reference in its entirety.

In some embodiments, the JAK1 inhibitor is a compound of Formula I

or a pharmaceutically acceptable salt thereof, wherein:

X is N or CH;

L is C(═O) or C(═O)NH;

A is phenyl, pyridinyl, or pyrimidinyl each of which is optionallysubstituted with 1 or 2 independently selected R¹ groups; and

each R¹ is, independently, fluoro, or trifluoromethyl.

In some embodiments, the compound of Formula I is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula I is4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[4-fluoro-2-(trifluoromethyl)phenyl]piperidine-1-carboxamide,or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula I is[3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-1-(1-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)azetidin-3-yl]acetonitrile,or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is a compound of Formula II

or a pharmaceutically acceptable salt thereof, wherein:

R² is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₆ cycloalkyl, or C₃₋₆cycloalkyl-C₁₋₃ alkyl, wherein said C₁₋₆ alkyl, C₃₋₆ cycloalkyl, andC₃₋₆ cycloalkyl-C₁₋₃ alkyl, are each optionally substituted with 1, 2,or 3 substituents independently selected from fluoro, —CF₃, and methyl;

R³ is H or methyl;

R⁴ is H, F, or Cl;

R⁵ is H or F;

R⁶ is H or F;

R⁷ is H or F;

R⁸ is H or methyl;

R⁹ is H or methyl;

R¹⁰ is H or methyl; and

R¹¹ is H or methyl.

In some embodiments, the compound of Formula II is4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide,or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is a compound of Formula III

or a pharmaceutically acceptable salt thereof, wherein:

Cy⁴ is a tetrahydro-2H-pyran ring, which is optionally substituted with1 or 2 groups independently selected from CN, OH, F, Cl, C₁₋₃ alkyl,C₁₋₃ haloalkyl, CN—C₁₋₃ alkyl, HO—C₁₋₃ alkyl, amino, C₁₋₃ alkylamino,and di(C₁₋₃ alkyl)amino, wherein said C₁₋₃ alkyl and di(C₁₋₃ alkyl)aminois optionally substituted with 1, 2, or 3 substituents independentlyselected from F, Cl, C₁₋₃ alkylaminosulfonyl, and C₁₋₃ alkylsulfonyl;and

R¹² is —CH₂—OH, —CH(CH₃)—OH, or —CH₂—NHSO₂CH₃.

In some embodiments, the compound of Formula III is((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile,or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is barcitinib,tofacitinib, oclacitinib, filgotinib, gandotinib, lestaurtinib,momelotinib, bacritinib, PF-04965842, upadacitinib, peficitinib,fedratinib, cucurbitacin I, ATI-501 (Aclaris), ATI-502 (Aclaris), JTE052(Leo Pharma and Japan Tobacco), or CHZ868.

In some embodiments, the inhibitor of JAK1 and/or JAK2 can be anisotopically-labeled compound, or a pharmaceutically acceptable saltthereof. An “isotopically” or “radio-labeled” compound is a compound ofthe disclosure where one or more atoms are replaced or substituted by anatom having an atomic mass or mass number different from the atomic massor mass number typically found in nature (i.e., naturally occurring).Suitable radionuclides that may be incorporated in compounds of thepresent disclosure include but are not limited to ²H (also written as Dfor deuterium), ³H (also written as T for tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N,¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I,¹²⁵I and ¹³¹I. For example, one or more hydrogen atoms in a compound ofthe present disclosure can be replaced by deuterium atoms, such as —CD₃being substituted for —CH₃).

One or more constituent atoms of the compounds described herein can bereplaced or substituted with isotopes of the atoms in natural ornon-natural abundance. In some embodiments, the compound includes atleast one deuterium atom. In some embodiments, the compound includes twoor more deuterium atoms. In some embodiments, the compound includes 1-2,1-3, 1-4, 1-5, or 1-6 deuterium atoms. In some embodiments, all of thehydrogen atoms in a compound can be replaced or substituted by deuteriumatoms.

Synthetic methods for including isotopes into organic compounds areknown in the art (Deuterium Labeling in Organic Chemistry by Alan F.Thomas (New York, N.Y., Appleton-Century-Crofts, 1971; The Renaissanceof H/D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and JochenZimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistryof Isotopic Labelling by James R. Hanson, Royal Society of Chemistry,2011). Isotopically labeled compounds can be used in various studiessuch as NMR spectroscopy, metabolism experiments, and/or assays.

Substitution with heavier isotopes, such as deuterium, may affordcertain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances. (seee.g., A. Kerekes et. al. J. Med. Chem. 2011, 54, 201-210; R. Xu et. al.J. Label Compd. Radiopharm. 2015, 58, 308-312). In particular,substitution at one or more metabolism sites may afford one or more ofthe therapeutic advantages.

Accordingly, in some embodiments, the inhibitor of JAK1 and/or JAK2 is acompound, wherein one or more hydrogen atoms in the compound arereplaced by deuterium atoms, or a pharmaceutically acceptable saltthereof.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is ruxolitinib,wherein one or more hydrogen atoms are replaced by deuterium atoms, or apharmaceutically acceptable salt thereof. In some embodiments, theinhibitor of JAK1 and/or JAK2 is any of the compounds in U.S. Pat. No.9,249,149 (which is incorporated herein by reference in its entirety),or a pharmaceutically acceptable salt thereof. In some embodiments, theinhibitor of JAK1 and/or JAK2 is CTP4-543, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the compound is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is selected from H and D;

each R² is independently selected from H and D, provided that each R²attached to a common carbon is the same;

each R³ is independently selected from H and D, provided that each R³attached to a common carbon is the same;

R⁴ is selected from H and D;

each R⁵ is the same and is selected from H and D; and

R⁶, R⁷, and R⁸ are each independently selected from H and D; providedthat when R¹ is H, each R² and each R³ are H, R⁴ is H, and each of R⁶,R⁷, and R⁸ is H, then each R⁵ is D.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is a compound ofFormula I selected from the following compounds 100-130 in the tablebelow (wherein R⁶, R⁷, and R⁸ are each H), or a pharmaceuticallyacceptable salt thereof. In some embodiments, the inhibitor of JAK1and/or JAK2 is a compound of Formula I selected from the followingcompounds 200-231 in the table below (wherein R⁶, R⁷, and R⁸ are eachD), or a pharmaceutically acceptable salt thereof.

Compound R¹ Each R² Each R³ R⁴ Each R⁵ 100 H H H D H 101 H H H H D 102 HH H D D 103 H H D H H 104 H H D D H 105 H H D H D 106 H H D D D 107 H DH H H 108 H D H D H 109 H D H H D 110 H D H D D 111 H D D H H 112 H D DD H 113 H D D H D 114 H D D D D 115 D H H H H 116 D H H D H 117 D H H HD 118 D H H D D 119 D H D H H 120 D H D D H 121 D H D H D 122 D H D D D123 D D H H H 124 D D H D H 125 D D H H D 126 D D H D D 127 D D D H H128 D D D D H 129 D D D H D 130 D D D D D 200 H H H D H 201 H H H H D202 H H H D D 203 H H D H H 204 H H D D H 205 H H D H D 206 H H D D D207 H D H H H 208 H D H D H 209 H D H H D 210 H D H D D 211 H D D H H212 H D D D H 213 H D D H D 214 H D D D D 215 D H H H H 216 D H H D H217 D H H H D 218 D H H D D 219 D H D H H 220 D H D D H 221 D H D H D222 D H D D D 223 D D H H H 224 D D H D H 225 D D H H D 226 D D H D D227 D D D H H 228 D D D D H 229 D D D H D 230 D D D D D 231 H H H H H

In some embodiments, the inhibitor of JAK1 and/or JAK2 is baricitinib,wherein one or more hydrogen atoms are replaced by deuterium atoms, or apharmaceutically acceptable salt thereof. In some embodiments, theinhibitor of JAK1 and/or JAK2 is any of the compounds in U.S. Pat. No.9,540,367 (which is incorporated herein by reference in its entirety),or a pharmaceutically acceptable salt thereof.

As used herein, the phrase “optionally substituted” means unsubstitutedor substituted. As used herein, the term “substituted” means that ahydrogen atom is removed and replaced by a substituent. It is to beunderstood that substitution at a given atom is limited by valency.

As used herein, the term “C_(n-m) alkyl”, employed alone or incombination with other terms, refers to a saturated hydrocarbon groupthat may be straight-chain or branched, having n to m carbon atoms. Insome embodiments, the alkyl group contains 1 to 6, or 1 to 3 carbonatoms. Examples of alkyl moieties include, but are not limited to,chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl,n-hexyl, 1,2,2-trimethylpropyl, and the like.

As used herein, the term “alkylene”, employed alone or in combinationwith other terms, refers to a divalent alkyl linking group, which can bebranched or straight-chain, where the two substituents may be attachedany position of the alkylene linking group. Examples of alkylene groupsinclude, but are not limited to, ethan-1,2-diyl, propan-1,3-diyl,propan-1,2-diyl, and the like.

As used herein, the term “HO—C₁₋₃-alkyl” refers to a group of formula-alkylene-OH, wherein said alkylene group has 1 to 3 carbon atoms.

As used herein, the term “CN—C₁₋₃ alkyl” refers to a C₁₋₃ alkylsubstituted by a cyano group.

As used herein, the term “amino” refers to a group of formula —NH₂.

As used herein, the term “di(C₁₋₃-alkyl)amino” refers to a group offormula —N(alkyl)₂, wherein the two alkyl groups each has,independently, 1 to 3 carbon atoms.

As used herein, the term “C₁₋₃ alkylamino” refers to a group of formula—NH(alkyl), wherein the alkyl group has 1 to 3 carbon atoms.

As used herein, the term “di(C₁₋₃ alkyl)aminosulfonyl” refers to a groupof formula —S(O)₂N(alkyl)₂, wherein each alkyl group independently has 1to 3 carbon atoms.

As used herein, the term “C₁₋₃ alkylsulfonyl” refers to a group offormula —S(O)₂-alkyl, wherein the alkyl group has 1 to 3 carbon atoms.

As used herein, “halo” or “halogen”, employed alone or in combinationwith other terms, includes fluoro, chloro, bromo, and iodo. In someembodiments, the halo group is fluoro or chloro.

As used herein, the term “C_(n-m) haloalkyl”, employed alone or incombination with other terms, refers to a C_(n-m) alkyl group having upto {2(n to m)+1} halogen atoms which may either be the same ordifferent. In some embodiments, the halogen atoms are fluoro atoms. Insome embodiments, the alkyl group has 1-6 or 1-3 carbon atoms. Examplehaloalkyl groups include CF₃, C₂F₅, CHF₂, CCl₃, CHCl₂, C₂Cl₅, and thelike. In some embodiments, the haloalkyl group is a fluoroalkyl group.

As used herein, the term “C₁₋₃ fluoroalkyl” refers to a C₁₋₃ alkyl groupthat may be partially or completely substituted by fluoro atoms.

As used herein, the term “C₃₋₆ cycloalkyl”, employed alone or incombination with other terms, refers to a non-aromatic monocyclichydrocarbon moiety, having 3-6 carbon atoms, which may optionallycontain one or more alkenylene groups as part of the ring structure. Oneor more ring-forming carbon atoms of a cycloalkyl group can be oxidizedto form carbonyl linkages. Exemplary C₃₋₆ cycloalkyl groups includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl,cyclohexenyl, cyclohexadienyl, and the like. In some embodiments, thecycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

As used herein, the term “C₃₋₆ cycloalkyl-C₁₋₃ alkyl” refers to a groupof formula —C₁₋₃ alkylene-C₃₋₆ cycloalkyl.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds thatcontain asymmetrically substituted carbon atoms can be isolated inoptically active or racemic forms. Methods on how to prepare opticallyactive forms from optically inactive starting materials are known in theart, such as by resolution of racemic mixtures or by stereoselectivesynthesis. Many geometric isomers of olefins, C═N double bonds, and thelike can also be present in the compounds described herein, and all suchstable isomers are contemplated in the present application. Cis andtrans geometric isomers of the compounds of the present application aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. In some embodiments, the compound has the(R)-configuration. In some embodiments, the compound has the(S)-configuration.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. An example method includes fractionalrecrystallization using a chiral resolving acid which is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, for example, optically activeacids, such as the D and L forms of tartaric acid, diacetyltartaricacid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid orthe various optically active camphorsulfonic acids such asβ-camphorsulfonic acid. Other resolving agents suitable for fractionalcrystallization methods include stereoisomerically pure forms ofα-methylbenzylamine (e.g., S and R forms, or diastereomerically pureforms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

Compounds described herein include tautomeric forms. Tautomeric formsresult from the swapping of a single bond with an adjacent double bondtogether with the concomitant migration of a proton. Tautomeric formsinclude prototropic tautomers which are isomeric protonation stateshaving the same empirical formula and total charge. Example prototropictautomers include ketone—enol pairs, amide—imidic acid pairs,lactam—lactim pairs, enamine—imine pairs, and annular forms where aproton can occupy two or more positions of a heterocyclic system, forexample, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be inequilibrium or sterically locked into one form by appropriatesubstitution. For example, it will be recognized that the followingpyrazole ring may form two tautomers:

It is intended that the claims cover both tautomers.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.hydrates and solvates) or can be isolated.

In some embodiments, the compounds described herein, or salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compounds describedherein. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compounds described herein, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The expressions, “ambient temperature” and “room temperature” or “rt” asused herein, are understood in the art, and refer generally to atemperature, e.g. a reaction temperature, that is about the temperatureof the room in which the reaction is carried out, for example, atemperature from about 20° C. to about 30° C.

The present application also includes pharmaceutically acceptable saltsof the compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present application include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The pharmaceutically acceptablesalts of the present application can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, non-aqueous media like ether, ethylacetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) oracetonitrile (ACN) are preferred. Lists of suitable salts are found inRemington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company,Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2(1977), each of which is incorporated herein by reference in itsentirety.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” a JAK with a compound of the invention includesthe administration of a compound of the present application to anindividual or patient, such as a human, having a JAK, as well as, forexample, introducing a compound of the invention into a samplecontaining a cellular or purified preparation containing the JAK.

As used herein, the term “subject”, “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans. In some embodiments,the “subject,” “individual,” or “patient” is in need of said treatment.

In some embodiments, the inhibitors are administered in atherapeutically effective amount. As used herein, the phrase“therapeutically effective amount” refers to the amount of activecompound or pharmaceutical agent that elicits the biological ormedicinal response that is being sought in a tissue, system, animal,individual or human by a researcher, veterinarian, medical doctor orother clinician.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) inhibiting the disease; for example, inhibiting a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., arresting further development of the pathology and/orsymptomatology); (2) ameliorating the disease; for example, amelioratinga disease, condition or disorder in an individual who is experiencing ordisplaying the pathology or symptomatology of the disease, condition ordisorder (i.e., reversing the pathology and/or symptomatology) such asdecreasing the severity of disease; or (3) preventing the disease,condition or disorder in an individual who may be predisposed to thedisease, condition or disorder but does not yet experience or displaythe pathology or symptomatology of the disease. In some embodiments,treating refers to inhibiting or ameliorating the disease. In someembodiments, treating is preventing the disease.

Combination Therapies

The methods described herein can further comprise administering one ormore additional therapeutic agents. The one or more additionaltherapeutic agents can be administered to a patient simultaneously orsequentially.

In some embodiments, the additional therapeutic agent is an antibiotic.In some embodiments, the antibiotic is clindamycin, doxycycline,minocycline, trimethoprim-sulfamethoxazole, erythromycin, metronidazole,rifampin, moxifloxacin, dapsone, or a combination thereof. In someembodiments, the antibiotic is clindamycin, doxycycline, minocycline,trimethoprim-sulfamethoxazole, or erythromycin in combination withmetronidazole. In some embodiments, the antibiotic is a combination ofrifampin, moxifloxacin, and metronidazole. In some embodiments, theantibiotic is a combination of moxifloxacin and rifampin.

In some embodiments, the additional therapeutic agent is a retinoid. Insome embodiments, the retinoid is etretinate, acitretin, orisotretinoin.

In some embodiments, the additional therapeutic agent is a steroid. Insome embodiments, the additional therapeutic agent is a corticosteroid.In some embodiments, the steroid is such as triamcinolone,dexamethasone, fluocinolone, cortisone, prednisone, prednisolone, orflumetholone.

In some embodiments, the additional therapeutic agent is ananti-TNF-alpha agent. In some embodiments, the anti-TNF-alpha agent isan anti-TNF-alpha antibody. In some embodiments, the anti-TNF-alphaagent is infliximab or etanercept, or adalimumab.

In some embodiments, the additional therapeutic agent is animmunosuppressant. In some embodiments, the immunosuppressant ismethotrexate or cyclosporin A. In some embodiments, theimmunosuppressant is mycophenolate mofetil or mycophenolate sodium.

In some embodiments, the additional therapeutic agent is finasteride,metformin, adapalene or azelaic acid.

In some embodiments, the method further comprises administering anadditional therapeutic agent selected from IMiDs, an anti-IL-6 agent, ahypomethylating agent, and a biologic response modifier (BRM).

Generally, a BRM is a substances made from living organisms to treatdisease, which may occur naturally in the body or may be made in thelaboratory. Examples of BRMs include IL-2, interferon, various types ofcolony-stimulating factors (CSF, GM-CSF, G-CSF), monoclonal antibodiessuch as abciximab, etanercept, infliximab, rituximab, trasturzumab, andhigh dose ascorbate.

In some embodiments, the hypomethylating agent is a DNAmethyltransferase inhibitor. In some embodiments, the DNAmethyltransferase inhibitor is selected from 5 azacytidine anddecitabine.

Generally, IMiDs are as immunomodulatory agents. In some embodiments,the IMiD is selected from thalidomide, lenalidomide, pomalidomide,CC-11006, and CC-10015.

In some embodiments, the method further comprises administering anadditional therapeutic agent selected from anti-thymocyte globulin,recombinant human granulocyte colony-stimulating factor (G CSF),granulocyte-monocyte CSF (GM-CSF), an erythropoiesis-stimulating agent(ESA), and cyclosporine.

In some embodiments, the method further comprises administering anadditional JAK inhibitor to the patient. In some embodiments, theadditional JAK inhibitor is barcitinib, tofacitinib, oclacitinib,filgotinib, gandotinib, lestaurtinib, momelotinib, bacritinib,PF-04965842, upadacitinib, peficitinib, fedratinib, cucurbitacin I, orCHZ868. One or more additional pharmaceutical agents such as, forexample, anti-inflammatory agents, immunosuppressants, as well as PI3Kδ,mTor, Bcr-Abl, Flt-3, RAF and FAK kinase inhibitors such as, forexample, those described in WO 2006/056399, which is incorporated hereinby reference in its entirety, or other agents can be used in combinationwith the compounds described herein for treatment of JAK-associateddiseases, disorders or conditions. The one or more additionalpharmaceutical agents can be administered to a patient simultaneously orsequentially.

Example Bcr-Abl inhibitors include the compounds, and pharmaceuticallyacceptable salts thereof, of the genera and species disclosed in U.S.Pat. No. 5,521,184, WO 04/005281, and U.S. Ser. No. 60/578,491, all ofwhich are incorporated herein by reference in their entirety.

Example suitable Flt-3 inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 03/037347, WO03/099771, and WO 04/046120, all of which are incorporated herein byreference in their entirety.

Example suitable RAF inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO05/028444, both of which are incorporated herein by reference in theirentirety.

Example suitable FAK inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 04/080980, WO04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402,all of which are incorporated herein by reference in their entirety.

In some embodiments, one or more of the compounds of the invention canbe used in combination with one or more other kinase inhibitorsincluding imatinib, particularly for treating patients resistant toimatinib or other kinase inhibitors.

In some embodiments, the additional therapeutic agent is fluocinoloneacetonide (Retisert®), or rimexolone (AL-2178, Vexol, Alcon).

In some embodiments, the additional therapeutic agent is cyclosporine(Restasis®).

In some embodiments, the additional therapeutic agent is selected fromDehydrex™ (Holles Labs), Civamide (Opko), sodium hyaluronate (Vismed,Lantibio/TRB Chemedia), cyclosporine (ST-603, Sirion Therapeutics),ARG101(T) (testosterone, Argentis), AGR1012(P) (Argentis), ecabet sodium(Senju-Ista), gefarnate (Santen), 15-(s)-hydroxyeicosatetraenoic acid(15(S)-HETE), cevilemine, doxycycline (ALTY-0501, Alacrity),minocycline, iDestrin™ (NP50301, Nascent Pharmaceuticals), cyclosporineA (Nova22007, Novagali), oxytetracycline (Duramycin, MOLI1901,Lantibio), CF101(2S,3S,4R,5R)-3,4-dihydroxy-5-[6-[(3-iodophenyl)methylamino]purin-9-yl]-N-methyl-oxolane-2-carbamyl,Can-Fite Biopharma), voclosporin (LX212 or LX214, Lux Biosciences),ARG103 (Agentis), RX-10045 (synthetic resolvin analog, Resolvyx), DYN15(Dyanmis Therapeutics), rivoglitazone (DE011, Daiichi Sanko), TB4(RegeneRx), OPH-01 (Ophtalmis Monaco), PCS101 (Pericor Science), REV1-31(Evolutec), Lacritin (Senju), rebamipide (Otsuka-Novartis), OT-551(Othera), PAI-2 (University of Pennsylvania and Temple University),pilocarpine, tacrolimus, pimecrolimus (AMS981, Novartis), loteprednoletabonate, rituximab, diquafosol tetrasodium (INS365, Inspire), KLS-0611(Kissei Pharmaceuticals), dehydroepiandrosterone, anakinra, efalizumab,mycophenolate sodium, etanercept (Embrel®), hydroxychloroquine, NGX267(TorreyPines Therapeutics), actemra, gemcitabine, oxaliplatin,L-asparaginase, or thalidomide.

In some embodiments, the additional therapeutic agent is ananti-angiogenic agent, cholinergic agonist, TRP-1 receptor modulator, acalcium channel blocker, a mucin secretagogue, MUC1 stimulant, acalcineurin inhibitor, a corticosteroid, a P2Y2 receptor agonist, amuscarinic receptor agonist, an mTOR inhibitor, another JAK inhibitor,Bcr-Abl kinase inhibitor, Flt-3 kinase inhibitor, RAF kinase inhibitor,and FAK kinase inhibitor such as, for example, those described in WO2006/056399, which is incorporated herein by reference in its entirety.In some embodiments, the additional therapeutic agent is a tetracyclinederivative (e.g., minocycline or doxycline). In some embodiments, theadditional therapeutic agent binds to FKBP12.

In some embodiments, the additional therapeutic agent is an alkylatingagent or DNA cross-linking agent; an anti-metabolite/demethylating agent(e.g., 5-flurouracil, capecitabine or azacitidine); an anti-hormonetherapy (e.g., hormone receptor antagonists, SERMs, or aromotaseinhibitor); a mitotic inhibitor (e.g. vincristine or paclitaxel); antopoisomerase (I or II) inhibitor (e.g. mitoxantrone and irinotecan); anapoptotic inducers (e.g. ABT-737); a nucleic acid therapy (e.g.antisense or RNAi); nuclear receptor ligands (e.g., agonists and/orantagonists: all-trans retinoic acid or bexarotene); epigenetictargeting agents such as histone deacetylase inhibitors (e.g.vorinostat), hypomethylating agents (e.g. decitabine); regulators ofprotein stability such as Hsp90 inhibitors, ubiquitin and/or ubiquitinlike conjugating or deconjugating molecules; or an EGFR inhibitor(erlotinib).

In some embodiments, the additional therapeutic agent includes anantibiotic, antiviral, antifungal, anesthetic, anti-inflammatory agentsincluding steroidal and non-steroidal anti-inflammatories, andanti-allergic agents. Examples of suitable medicaments includeaminoglycosides such as amikacin, gentamycin, tobramycin, streptomycin,netilmycin, and kanamycin; fluoroquinolones such as ciprofloxacin,norfloxacin, ofloxacin, trovafloxacin, lomefloxacin, levofloxacin, andenoxacin; naphthyridine; sulfonamides; polymyxin; chloramphenicol;neomycin; paramomycin; colistimethate; bacitracin; vancomycin;tetracyclines; rifampin and its derivatives (“rifampins”); cycloserine;beta-lactams; cephalosporins; amphotericins; fluconazole; flucytosine;natamycin; miconazole; ketoconazole; corticosteroids; diclofenac;flurbiprofen; ketorolac; suprofen; cromolyn; lodoxamide; levocabastin;naphazoline; antazoline; pheniramine; or azalide antibiotic.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds of the invention can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical (includingtransdermal, epidermal, ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer;intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.

In some embodiments, the administration is topical. In some embodiments,the administration is topical administration to the skin.

In some embodiments, the administration is oral.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, the compound of the invention or apharmaceutically acceptable salt thereof, in combination with one ormore pharmaceutically acceptable carriers (excipients). In someembodiments, the composition is suitable for topical administration. Inmaking the compositions of the invention, the active ingredient istypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, for example, a capsule, sachet,paper, or other container. When the excipient serves as a diluent, itcan be a solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

The compounds of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the compounds of the invention can beprepared by processes known in the art, e.g., see International App. No.WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

In some embodiments, the pharmaceutical composition comprises silicifiedmicrocrystalline cellulose (SMCC) and at least one compound describedherein, or a pharmaceutically acceptable salt thereof. In someembodiments, the silicified microcrystalline cellulose comprises about98% microcrystalline cellulose and about 2% silicon dioxide w/w.

In some embodiments, the composition is a sustained release compositioncomprising at least one compound described herein, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier. In some embodiments, the composition comprises at least onecompound described herein, or a pharmaceutically acceptable saltthereof, and at least one component selected from microcrystallinecellulose, lactose monohydrate, hydroxypropyl methylcellulose, andpolyethylene oxide.

In some embodiments, the composition comprises at least one compounddescribed herein, or a pharmaceutically acceptable salt thereof, andmicrocrystalline cellulose, lactose monohydrate, and hydroxypropylmethylcellulose. In some embodiments, the composition comprises at leastone compound described herein, or a pharmaceutically acceptable saltthereof, and microcrystalline cellulose, lactose monohydrate, andpolyethylene oxide. In some embodiments, the composition furthercomprises magnesium stearate or silicon dioxide. In some embodiments,the microcrystalline cellulose is Avicel PH102™. In some embodiments,the lactose monohydrate is Fast-flo 316™. In some embodiments, thehydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208 K4M(e.g., Methocel K4 M Premier™) and/or hydroxypropyl methylcellulose 2208K100LV (e.g., Methocel KOOLV™). In some embodiments, the polyethyleneoxide is polyethylene oxide WSR 1105 (e.g., Polyox WSR 1105™).

In some embodiments, a wet granulation process is used to produce thecomposition. In some embodiments, a dry granulation process is used toproduce the composition.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 1 to about 1,000 mg, from about 1 mg to about 100mg, from 1 mg to about 50 mg, and from about 1 mg to 10 mg of activeingredient. Preferably, the dosage is from about 1 mg to about 50 mg orabout 1 mg to about 10 mg of active ingredient. In some embodiments,each dosage contains about 10 mg of the active ingredient. In someembodiments, each dosage contains about 50 mg of the active ingredient.In some embodiments, each dosage contains about 25 mg of the activeingredient. The term “unit dosage forms” refers to physically discreteunits suitable as unitary dosages for human subjects and other mammals,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect, in associationwith a suitable pharmaceutical excipient.

In some embodiments, the compositions comprise from about 1 to about1,000 mg, from about 1 mg to about 100 mg, from 1 mg to about 50 mg, andfrom about 1 mg to 10 mg of active ingredient. Preferably, thecompositions comprise from about 1 mg to about 50 mg or about 1 mg toabout 10 mg of active ingredient. One having ordinary skill in the artwill appreciate that this embodies compounds or compositions containingabout 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg toabout 25 mg, about 1 mg to about 50 mg of the active ingredient.

In some embodiments, the dosage of the compound, or a pharmaceuticallyacceptable salt thereof, is 15, 30, 60 or 90 mg on a free base basis. Insome embodiments, the dosage is 15, 30, 60 or 90 mg on a free basebasis, of Compound 4, or a pharmaceutically acceptable salt thereof. Insome embodiments, the dosage of the compound, or a pharmaceuticallyacceptable salt thereof, is 15 mg on a free base basis. In someembodiments, the dosage of the compound, or a pharmaceuticallyacceptable salt thereof, is 30 mg on a free base basis. In someembodiments, the dosage of the compound, or a pharmaceuticallyacceptable salt thereof, is 60 mg on a free base basis. In someembodiments, the dosage of the compound, or a pharmaceuticallyacceptable salt thereof, is 90 mg on a free base basis.

The active compound may be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present application. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present application.

The tablets or pills of the present application can be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by anenteric layer which serves to resist disintegration in the stomach andpermit the inner component to pass intact into the duodenum or to bedelayed in release. A variety of materials can be used for such entericlayers or coatings, such materials including a number of polymeric acidsand mixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentapplication can be incorporated for administration orally or byinjection include aqueous solutions, suitably flavored syrups, aqueousor oil suspensions, and flavored emulsions with edible oils such ascottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in can be nebulized by use of inert gases. Nebulizedsolutions may be breathed directly from the nebulizing device or thenebulizing device can be attached to a face masks tent, or intermittentpositive pressure breathing machine. Solution, suspension, or powdercompositions can be administered orally or nasally from devices whichdeliver the formulation in an appropriate manner.

Topical formulations can contain one or more conventional carriers. Insome embodiments, ointments can contain water and one or morehydrophobic carriers selected from, for example, liquid paraffin,polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and thelike. Carrier compositions of creams can be based on water incombination with glycerol and one or more other components, e.g.glycerinemonostearate, PEG-glycerinemonostearate and cetylstearylalcohol. Gels can be formulated using isopropyl alcohol and water,suitably in combination with other components such as, for example,glycerol, hydroxyethyl cellulose, and the like. In some embodiments,topical formulations contain at least about 0.1, at least about 0.25, atleast about 0.5, at least about 1, at least about 2, or at least about 5wt % of the compound of the invention. The topical formulations can besuitably packaged in tubes of, for example, 100 g which are optionallyassociated with instructions for the treatment of the select indication,e.g., psoriasis or other skin condition.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of a compound of the present application can varyaccording to, for example, the particular use for which the treatment ismade, the manner of administration of the compound, the health andcondition of the patient, and the judgment of the prescribing physician.The proportion or concentration of a compound of the invention in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the compounds of the inventioncan be provided in an aqueous physiological buffer solution containingabout 0.1 to about 10% w/v of the compound for parenteraladministration. Some typical dose ranges are from about 1 μg/kg to about1 g/kg of body weight per day. In some embodiments, the dose range isfrom about 0.01 mg/kg to about 100 mg/kg of body weight per day. Thedosage is likely to depend on such variables as the type and extent ofprogression of the disease or disorder, the overall health status of theparticular patient, the relative biological efficacy of the compoundselected, formulation of the excipient, and its route of administration.Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

The compositions of the invention can further include one or moreadditional pharmaceutical agents, examples of which are listedhereinabove.

Kits

The present application also includes pharmaceutical kits useful, forexample, in the treatment and/or prevention of cytokine-related diseasesor disorders, such as CRS, which include one or more containerscontaining a pharmaceutical composition comprising a therapeuticallyeffective amount of a compound described herein. Such kits can furtherinclude, if desired, one or more of various conventional pharmaceuticalkit components, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

EXAMPLES

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results.

Example A: In Vitro JAK Kinase Assay

JAK1 inhibitors that can be used for the treatment of cytokine-relateddiseases or disorders are tested for inhibitory activity of JAK targetsaccording to the following in vitro assay described in Park et al.,Analytical Biochemistry 1999, 269, 94-104. The catalytic domains ofhuman JAK1 (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3 (a.a.781-1124) with an N-terminal His tag are expressed using baculovirus ininsect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3was assayed by measuring the phosphorylation of a biotinylated peptide.The phosphorylated peptide was detected by homogenous time resolvedfluorescence (HTRF). ICsos of compounds are measured for each kinase inthe 40 microL reactions that contain the enzyme, ATP and 500 nM peptidein 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL(0.01%) BSA. For the 1 mM IC₅₀ measurements, ATP concentration in thereactions is 1 mM. Reactions are carried out at room temperature for 1hour and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europiumlabeled antibody takes place for 40 minutes and HTRF signal was measuredon a Fusion plate reader (Perkin Elmer, Boston, Mass.). The compounds inTable 1 were tested in this assay and shown to have the IC₅₀ values inTable 1

Example B: Safety and Efficacy Study of JAK1 and/or JAK2 Inhibitors inSubjects with Moderate to Severe Hidradenitis Suppurativa

A randomized, double-blind, placebo-control, multicenter study isconducted on men and women aged 18-75 years with moderate (Hurley StageII) to severe (Hurley Stage III) hidradenitis suppurativa for at least 6months. Hurley stage I is associated with abscess formation (single ormultiple) without sinus tracts and cicatrization. Hurley stage II isassociated with recurrent abscesses with tract formation andcicatrization; single or multiple, widely separated lesions. Hurleystage III is associated with diffuse or near-diffuse involvement ormultiple interconnected tracts and abscesses across the entire area.Study participants are randomized into 5 groups (about 50 participantsper group) and treated with either 15, 30, 60 or 90 mg of an inhibitorof JAK1 and/or JAK2 (e.g., ruxolitinib, Compound 4, or Compound 5, or apharmaceutically acceptable salt thereof), or placebo. At week 16(primary endpoint), participants in the placebo group are re-randomizedequally to active treatment arms for 8 weeks. The blind is maintained.The primary endpoint is the proportion of subjects achievingHidradenitis Suppurativa Clinical Response (HiSCR) at week 16.

Secondary endpoints include (1) Proportion of subjects with HiSCR overbaseline at each visit; (2) Proportion of subjects achieving abscess andinflammatory nodule (AN) count of 0 to 2 at each visit; (3) Mean changefrom baseline in HS Pain Numeric Rating Scalel) at each visit; (4)Change in modified Sartorius scale at week 16 and week 24; (5) Change innumber of draining fistulas count at each visit; (6) Proportion ofsubjects requiring lesional rescue treatment through week 24; (7) Numberof episodes of lesional rescue treatments through week 24; (8)Population PK of the inhibitor of JAK1 and/or JAK2 (e.g., apparentclearance, apparent volume of distribution); (9) Safety and tolerabilityassessed by monitoring the frequency, duration, and severity of AEs,physical examination, vital signs, and laboratory data for hematology,serum chemistry, and urinalysis; (10) Change in Dermatology Quality ofLife Index (DLQI) assessment; (11) Change in the severity of the diseasefrom baseline as assessed by the IHS43 scoring at each visit; (12)Change in hidradenitis suppurativa quality of life (HiSQOL) assessmentat each visit over baseline; and (13) Assessment of thedose/exposure-response on percentage change from baseline in terms ofefficacy and safety endpoints during the treatment periods.

HiSCR is defined as at least a 50% reduction in abscess and inflammatorynodule (AN) count with no increase in abscess count and no increase indraining fistula count at week 16 relative to baseline). Pain NumericRating Scale is used to assess the worst skin and average skin pain dueto HS. Ratings for the 2 items range from 0 (no skin pain) to 10 (skinpain as bad as you can imagine). The assessments are recorded on a dailydiary by participants before they go to bed and based on a recall periodof the “last 24 hours.” The modified Sartorius Scale is used to quantifythe severity of HS. Points are awarded for 12 body areas (left and rightaxilla, left and right sub/inframammary areas, intermammary area, leftand right buttocks, left and right inguino-crural folds, perianal area,perineal area, and other): points awarded for nodule (2 points foreach); abscesses (4 points); fistulas (4 points); scar (1 point); andlongest distance between two lesions (2-6 points, 0 if no lesions); andif lesions are separated buy normal skin (yes-0 point; no-6 points). Thetotal Sartorius Scale is the sum of the 12 regional scores. Lesionalrescue treatment: In the event that an acutely painful lesion requiresan immediate intervention, physicians have the option to perform rescueinterventions. Only two types of interventions are allowed: (1)injection with intralesional triamcinolone acetonide suspension (up to30 mg in total at the same visit) and/or (2) incision and drainage. Anintervention can occur on maximally two different lesions at the samevisit or on the same lesion at two different study visits. The samelesion cannot be treated two times at the same visit. If a subjectrequires more than two interventions before week 16, then they arediscontinued from the study. International Hidradenitis SuppurativaSeverity Score System (IHS4): 1H4 (points)=(number of nodules×1)+(numberof abscesses×2)+(number of draining tunnels [fistulae/sinuses]×4). MildHS: ≤3 points; Moderate HS: 4-10 points; Severe HS: ≥11 points.

Study treatment 1 (Active) includes an oral tablet containing 15 mg of4-[3-(Cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.Dosing levels include 15 mg (1 tablet), 30 mg (2 tablets), 60 mg (4tablets) and 90 mg (6 tablets). Study treatment 2 (Placebo) includes anoral tablet placebo.

Blood samples for measurement of plasma concentrations of the inhibitorof JAK1 and/or JAK2 are taken, at least, on weeks 2, 12, 16, 20 and 24before and after administration of study drug at predose, 1 hourpostdose and 2-5 hours post dose time points. At the prematurediscontinuation visit if the subjects discontinues prior to week 8, atrough PK sample is collected if feasible. The date/time of the lastprior dose administration is also be recorded.

Superiority tests of the inhibitor of JAK1 and/or JAK2 at 90, 60, 30 and15 mg compared with placebo is carried out using the Hochberg procedureat an overall 2-sided α=0.05 level. Comparisons between each activegroup and placebo at week 16 is performed with a logistic regression. Atall dose levels the superiority tests are significant (for example, a10%, 20%, 30%, 40%, or 50% improvement in HiSCR (HidradenitisSuppurativa Clinical Response)) and demonstrate the efficacy of theinhibitor of JAK1 and/or JAK2 to treat HS. The tests show a reduction innodules and non-inferiority/superiority compared to placebo.

All secondary and exploratory efficacy measures are evaluated usingdescriptive statistics. The clinical safety data (vital signs, routinelaboratory tests, and AEs) are analyzed using descriptive statistics.Exposure-response (E-R) relationship(s) between plasma JAK1 and/or JAK2inhibitor PK exposures and efficacy/safety data are determined. Aninterim analysis to estimate treatment response and facilitate planningfor future studies is conducted when at least half of the randomizedsubjects reach week 16.

Example C. Interferon-Gamma and Tumor Necrosis-Alpha Induced JanusKinase Expression in Keratinocyte and Subsequent Production ofInflammatory Mediators

Transformed human keratinocyte (HaCaT) cells were purchased fromAddexBio (Catalog # T0020001) and cultured in Optimized Dulbecco'sModified Eagle's Medium (AddexBio, Catalog # C0003-02) supplemented with10% Fetal Bovine Serum (Hyclone, Catalog #16140-071) and 1×Penicillin/Streptomycin (Gibco, Catalog #15140-122). When cells reached80-90% confluency they were washed with 1×DPBS then detached from tissueculture flasks by incubation with 0.25% Trypsin (Gibco, Catalog#25200-056) for 3-5 minutes at 37° C./5% CO₂. Cell culture media wasadded to trypsinized cells then cell suspension was transferred to asterile 15 mL centrifuge tube to be spun down for 10 minutes at 1300rpms. Media containing trypsin was aspirated from the cell pellet andthen the pellet was re-suspended in 10 mL of cell culture media. Cellswere counted using a Countess II automated cell counter then seeded intotissue culture treated 24 well plates at a concentration of 4×10⁴cells/mL and incubated for 48 hours at 37° C./5% CO₂. After 48 hoursmedia was removed and replaced with 500 uL of either cell culture mediaor a combinatory stimulation of Recombinant Human Interferon gamma (R&DSystems, Catalog #285-IF-100) and Recombinant Human Tumor NecrosisFactor alpha (R&D Systems, Catalog #210-TA-020). HaCaT cells treatedwith the combinatory cytokine stimulation were treated at finalconcentrations of 10 ng/mL, 25 ng/mL, 50 ng/mL, or 100 ng/mL of eachcytokine. Treated plates were mixed by gentle agitation for 30 secondsthen incubated for 24 hours at 37° C./5% CO₂. At the end of the 24 hourincubation, media was immediately removed from each plate.

RNA was isolated from HaCaT cells using the QuantiGene Plex Assayreagents and protocols (Affymetrix, Catalog # QGP-232-M18042302). Cellswere washed with 1×DPBS then lysed by incubation with providedQuantiGene lysis buffer for 30 minutes at 50-55° C. Cell lysates wereincubated for 18-24 hours at 55° C. with capture beads and probe setdesigned to specifically hybridize to mRNA from targets of interest. Thepanel of 32 targets of interest included housekeeping genes used for thenormalization of the results. After the 18-24 hour incubation signal wasamplified utilizing branched DNA methodologies, according to themanufacturer's procedures (Affymetrix, Catalog # QGP-232-M18042302).After hybridization and wash steps assay plate was read on the Luminex200 and data were expressed as Net Median Fluorescence Intensity. Datawas then normalized to the Net Median Fluorescence Intensity of thehousekeeping gene HPRT1 (Table 2).

TABLE 2 Stimulation of Human Keratinocytes with TNFα and IFNγ Inducesthe JAK/STAT Pathway and Pro-Inflammatory Cytokines Gene TreatmentMFI^(a) p-value JAK1 Vehicle 126.7 ± 6.55  —  10 ng/ml TNFα/IFNγ 178.19± 3.41  <.0001  25 ng/ml TNFα/IFNγ 195.02 ± 3.47  <.0001  50 ng/mlTNFα/IFNγ 198.23 ± 2.52  <.0001 100 ng/ml TNFα/IFNγ 207.34 ± 3.91 <.0001 JAK2 Vehicle 21.7 ± 0.53 —  10 ng/ml TNFα/IFNγ 154.13 ± 11.65 <.0001  25 ng/ml TNFα/IFNγ 174.07 ± 12.34  <.0001  50 ng/ml TNFα/IFNγ180.71 ± 13.63  <.0001 100 ng/ml TNFα/IFNγ 187.94 ± 13.12  <.0001 JAK3Vehicle  0.1 ± 0.02 —  10 ng/ml TNFα/IFNγ 0.16 ± 0.05 0.8111  25 ng/mlTNFα/IFNγ 0.18 ± 0.05 0.596  50 ng/ml TNFα/IFNγ 0.33 ± 0.06 0.0082 100ng/ml TNFα/IFNγ 0.28 ± 0.06 0.0532 TYK2 Vehicle 167.84 ± 2.25  —  10ng/ml TNFα/IFNγ 240.49 ± 4.4   <.0001  25 ng/ml TNFα/IFNγ 250.15 ± 3.41 <.0001  50 ng/ml TNFα/IFNγ 257.24 ± 3.55  <.0001 100 ng/ml TNFα/IFNγ265.37 ± 3.1   <.0001 STAT1 Vehicle 484.33 ± 4.52  —  10 ng/ml TNFα/IFNγ3834.09 ± 65.62  <.0001  25 ng/ml TNFα/IFNγ 3935.51 ± 66.15  <.0001  50ng/ml TNFα/IFNγ 3943.03 ± 63.05  <.0001 100 ng/ml TNFα/IFNγ 4136.09 ±67.06  <.0001 STAT3 Vehicle 606.76 ± 11.51  —  10 ng/ml TNFα/IFNγ1561.14 ± 40.35  <.0001  25 ng/ml TNFα/IFNγ 1652.97 ± 39.53  <.0001  50ng/ml TNFα/IFNγ 1666.52 ± 52.15  <.0001 100 ng/ml TNFα/IFNγ 1742.81 ±38.26  <.0001 STAT4 Vehicle 2.27 ± 0.12 —  10 ng/ml TNFα/IFNγ 3.78 ±0.22 <.0001  25 ng/ml TNFα/IFNγ 3.84 ± 0.23 <.0001  50 ng/ml TNFα/IFNγ3.72 ± 0.25 <.0001 100 ng/ml TNFα/IFNγ 3.61 ± 0.28 0.0003 STAT5A Vehicle1.03 ± 0.1  —  10 ng/ml TNFα/IFNγ 26.06 ± 3.1  <.0001  25 ng/mlTNFα/IFNγ 28.58 ± 3.23  <.0001  50 ng/ml TNFα/IFNγ 31.01 ± 3.37  <.0001100 ng/ml TNFα/IFNγ 29.61 ± 2.91  <.0001 STAT6 Vehicle 626.95 ± 22    — 10 ng/ml TNFα/IFNγ 1010.38 ± 14.28  <.0001  25 ng/ml TNFα/IFNγ 1044.97± 12.71  <.0001  50 ng/ml TNFα/IFNγ 1039.59 ± 10.5   <.0001 100 ng/mlTNFα/IFNγ 1059.01 ± 13.45  <.0001 IL1A Vehicle 156.9 ± 1.89  —  10 ng/mlTNFα/IFNγ 1786.44 ± 31.13  <.0001  25 ng/ml TNFα/IFNγ 2135.03 ± 66.58 <.0001  50 ng/ml TNFα/IFNγ 2256.89 ± 90.79  <.0001 100 ng/ml TNFα/IFNγ2459.6 ± 106.2  <.0001 IL6 Vehicle 5.89 ± 0.19 —  10 ng/ml TNFα/IFNγ311.31 ± 38.81  0.0002  25 ng/ml TNFα/IFNγ 410.93 ± 52.93  <.0001  50ng/ml TNFα/IFNγ 464.27 ± 61.46  <.0001 100 ng/ml TNFα/IFNγ 519.31 ±68.04  <.0001 ^(a)Data are presented as the mean ± standard error (SEM)

Target proteins of interest in the media were detected and quantifiedusing the ProCarta Multiplex Immunoassay reagents and protocols(Invitrogen, Catalog #EPX450-12171-901). Media was incubated withantibody conjugated beads designed to bind to the epitopes of specifictarget proteins and identify the bound protein through the bead'sdistinctive spectral pattern. Biotinylated detection antibodies,designed to bind to different epitopes of the same target proteins, andStreptavidin-PE are added to assay plates to quantify the amount of thetarget protein. Assay plates were read on the Luminex 200 and data wereexpressed as Net Median Fluorescence Intensity. The Net MedianFluorescence Intensity values for the antigen standard curve, preparedaccording to the manufacturer's procedures (Invitrogen, Catalog #EPX450-12171-901) were plotted against the expected concentrations foreach standard. The concentration of each protein was extrapolated fromthe antigen standard curve and concentrations were expressed as pg/mL(Table 3).

TABLE 3 Stimulation of Human Keratinocytes with TNFα and IFNγ Inducesthe Pro-Inflammatory Cytokine Production Protein Treatment pg/mL^(a)p-value IL-1α Vehicle 0.37 ± 0.05 —  10 ng/ml TNFα/IFNγ 13.22 ± 1.24 <.0001  25 ng/ml TNFα/IFNγ 15.12 ± 1.48  <.0001  50 ng/ml TNFα/IFNγ14.74 ± 1.45  <.0001 100 ng/ml TNFα/IFNγ 13.64 ± 1.29  <.0001 IL-6Vehicle 72.86 ± 9.77  —  10 ng/ml TNFα/IFNγ 2012.1 ± 337.23 0.0001  25ng/ml TNFα/IFNγ 2329.01 ± 384.78  <.0001  50 ng/ml TNFα/IFNγ 2208.6 ±370.81 <.0001 100 ng/ml TNFα/IFNγ 1889.75 ± 298.39  0.0004 IP-10 Vehicle16.61 ± 1.6  —  10 ng/ml TNFα/IFNγ 3275.51 ± 174.48  <.0001  25 ng/mlTNFα/IFNγ 3243.28 ± 178.41  <.0001  50 ng/ml TNFα/IFNγ 3209.56 ± 211.43 <.0001 100 ng/ml TNFα/IFNγ 2978.45 ± 167.27  <.0001 MIP1α Vehicle 7.47 ±1.13 —  10 ng/ml TNFα/IFNγ 525.75 ± 87.5  <.0001  25 ng/ml TNFα/IFNγ546.69 ± 92.35  <.0001  50 ng/ml TNFα/IFNγ 531.55 ± 91.88  <.0001 100ng/ml TNFα/IFNγ 409.14 ± 60.62  0.0012 RANTES Vehicle 11.78 ± 1.41  — 10 ng/ml TNFα/IFNγ 126.13 ± 5.15  <.0001  25 ng/ml TNFα/IFNγ 127.73 ±2.8   <.0001  50 ng/ml TNFα/IFNγ 119.95 ± 4.67  <.0001 100 ng/mlTNFα/IFNγ 103.48 ± 7.09  <.0001 ^(a)Data are presented as the mean ±standard error (SEM)

Example D. Janus Kinase Inhibitors Interfere with Interferon-Gamma andTumor Necrosis-Alpha Mediated Inflammation in Keratinocytes

Transformed human keratinocyte (HaCaT) cells were purchased fromAddexBio (Catalog # T0020001) and cultured as outlined in Example C.Four compounds A-D (A: ruxolitinib, B: itacitinib({1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile),C: 4-[3-(Cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide,D:((2R,5S)-5-{2-[(1R)-1-Hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile)were reconstituted in DMSO then each compound was serial diluted withcell culture media to 400 nM, 200 nM, 100 nM, and 50 nM concentrations.After 48 hours, cell culture media was removed from 24 well plates andreplaced with 250 uL of media containing serial diluted drug, thenincubated for 15 minutes at 37° C./5% CO₂. After drug incubation, 250 uLof combinatory stimulation containing Recombinant Human Interferon gamma(R&D Systems, Catalog #285-IF-100) and Recombinant Human Tumor NecrosisFactor alpha (R&D Systems, Catalog #210-TA-020) was added to plates. Thefinal concentration of Recombinant Human Interferon gamma andRecombinant Human Tumor Necrosis Factor alpha was 25 ng/mL of eachcytokine. Cytokine stimulation added to wells containing drug broughtthe final concentrations for each drug treatment to 25 nM, 50 nM, 100nM, and 200 nM. Treated plates were mixed by gentle agitation for 30seconds then incubated for 24 hours at 37° C./5% CO₂. At the end of the24 hour incubation media was immediately removed from each plate.

RNA was isolated from HaCaT cells using the QuantiGene Plex Assayreagents and protocols (Affymetrix, Catalog # QGP-232-M18042302)according to the manufacturer's guidelines. Cells were washed with1×DPBS then lysed by incubation with provided QuantiGene lysis bufferfor 30 minutes at 50-55° C. Cell lysates were incubated for 18-24 hoursat 55° C. with capture beads and probe set designed to specificallyhybridize to mRNA from targets of interest. Genes included housekeepinggenes (eg. HPRT1, GAPDH) used for the normalization of the results.After the 18-24 hour incubation signal was amplified utilizing branchedDNA methodologies, according to the manufacturer's procedures(Affymetrix, Catalog # QGP-232-M18042302). After hybridization and washsteps assay plate was read on the Luminex 200 and data were expressed asNet Median Fluorescence Intensity. Data was then normalized to the NetMedian Fluorescence Intensity of the housekeeping gene HPRT1 (Table 4).

TABLE 4 Normalized Expression of Target Genes in Human Keratinocytecells Stimulated with TNFα and IFNγ in the Presence/Absence of JAKInhibitors Drug Compound A Compound B Compound C Compound D Stimu-Concen- p- p- p- p- Gene lation^(a) tration MFI^(b) value^(c) MFI^(b)value^(c) MFI^(b) value^(c) MFI^(b) value^(c) JAK1 — — 183.21 ± 7.55 25ng/mL — 213.93 ± 5.55^(€) — 200 nM 159.13 ± 7.08  — 171.53 ± 9.49  —177.67 ± 11.84 — 177.97 ± 14.91 — 25 ng/mL  25 nM 206.18 ± 7.99  0.894216.23 ± 6.41  0.9993 206.29 ± 6.84  0.7834 200.4 ± 9.84 0.5654 25 ng/mL 50 nM 195.48 ± 9.54  0.2925 210.42 ± 10.89 0.9965 194.2 ± 8.24 0.0852210.52 ± 7.73  0.9942 25 ng/mL 100 nM 186.97 ± 7.49  0.0621 205.03 ±11.49 0.9026 193.28 ± 4.55  0.0669 200.25 ± 8.15  0.5562 25 ng/mL 200 nM180.99 ± 8.58  0.0191 195.97 ± 10.45 0.4597 182.86 ± 4.07  0.0026 190.53± 7.68  0.1286 JAK2 — — 25.35 ± 0.95 25 ng/mL — 126.63 ± 4.89^(¥) — 200nM 23.67 ± 0.92 — 25.21 ± 1.12 — 25.25 ± 1.04 — 25.67 ± 1.03 — 25 ng/mL 25 nM  89.4 ± 2.21 <.0001 109.39 ± 2.8   0.0021 114.94 ± 2.16  0.0419108.89 ± 3.25  0.0165 25 ng/mL  50 nM  69.7 ± 1.78 <.0001   101 ± 2.26<.0001 107.16 ± 2.86  0.0003 106.83 ± 5.94  0.0063 25 ng/mL 100 nM 54.4± 1.8 <.0001  94.5 ± 2.65 <.0001 95.51 ± 3.13 <.0001 102.64 ± 3.52 0.0007 25 ng/mL 200 nM 40.25 ± 1.3  <.0001 89.16 ± 3.43 <.0001 91.17 ±2.15 <.0001 92.21 ± 2.9  <.0001 JAK3 — — 0.66 ± 0.14 25 ng/mL — 0.52 ±0.16 — 200 nM  0.53 ± 0.09 —  0.63 ± 0.10 —  0.68 ± 0.17 —  0.71 ± 0.15— 25 ng/mL  25 nM  0.81 ± 0.15 0.5284  0.84 ± 0.12 0.3247  1.02 ± 0.190.1022  0.97 ± 0.18 0.2187 25 ng/mL  50 nM  1.01 ± 0.23 0.1284  0.83 ±0.15 0.3497  0.99 ± 0.16 0.1493  0.99 ± 0.20 0.1854 25 ng/mL 100 nM 0.84 ± 0.13 0.4473  0.92 ± 0.13 0.1608  0.99 ± 0.15 0.1491  1.01 ± 0.170.1531 25 ng/mL 200 nM  0.68 ± 0.13 0.9133  0.79 ± 0.15 0.4876  0.85 ±0.15 0.4323  0.86 ± 0.16 0.4442 TYK2 — — 217.40 ± 8.13 25 ng/mL — 296.98± 6.92^(¥) — 200 nM 205.57 ± 10.87 — 217.28 ± 10.09 — 217.28 ± 14.28 —220.78 ± 12.01 — 25 ng/mL  25 nM 298.27 ± 10.83 >0.999 292.92 ± 7.99 0.9929 283.97 ± 8.59  0.5015 283.93 ± 8.16  0.7981 25 ng/mL  50 nM287.93 ± 16.28 0.9305 287.31 ± 11.08 0.8603 273.68 ± 7.44  0.0823 307.36± 14.87 0.8958 25 ng/mL 100 nM 260.21 ± 7.05  0.0546 284.15 ± 9.62 0.7043   266 ± 6.82 0.0123 280.63 ± 10.46 0.65 25 ng/mL 200 nM 264.75 ±8.44  0.1204 277.52 ± 8.67  0.3578 263.49 ± 5.05  0.0061 283.28 ± 10.880.7707 STAT1 — — 545.83 ± 15.37 25 ng/mL — 3106.13 ± 217.15^(¥) — 200 nM526.90 ± 13.46 — 535.07 ± 22.13 — 554.39 ± 11.80 — 554.64 ± 11.36 — 25ng/mL  25 nM 2907.12 ± 206.85 0.8632 2868.69 ± 202.69 0.7833 3111.17 ±182.20 >0.9999 3164.74 ± 242.35 0.9986 25 ng/mL  50 nM 2902.82 ± 173.710.8544 2862.58 ± 163.98 0.7685 3058.64 ± 154.86 0.9989 3017.08 ± 167.960.9928 25 ng/mL 100 nM 2712.93 ± 182.91 0.3789 2790.32 ± 176.4  0.58073035.33 ± 122.14 0.995 2999.87 ± 197.86 0.9862 25 ng/mL 200 nM 2475.58 ±134.64 0.0734  2857.2 ± 174.57 0.7553 2984.14 ± 163.4  0.9634 3161.66 ±135.8  0.9988 STAT3 — — 751.20 ± 14.97 25 ng/mL — 1608.39 ± 70.09^(¥) —200 nM 728.97 ± 20.48 — 732.19 ± 23.03 — 746.17 ± 16.73 — 750.90 ± 27.68— 25 ng/mL  25 nM 1434.08 ± 43.26  0.074 1466.73 ± 66.75  0.3206 1557.84± 58.15  0.9399 1572.76 ± 65.5   0.988 25 ng/mL  50 nM 1301.55 ± 51.7  0.0005 1437.28 ± 60.69  0.1762 1519.61 ± 69.92  0.7044 1543.4 ± 58.650.9042 25 ng/mL 100 nM 1150.46 ± 52.66  <.0001 1373.34 ± 55.51  0.03521457.24 ± 54.48  0.26 1549.17 ± 89.41  0.9288 25 ng/mL 200 nM 1082.84 ±39.32  <.0001 1400.77 ± 58.44  0.0738 1483.1 ± 51.73 0.4201 1570.19 ±51.51  0.9845 STAT4 — — 4.52 ± 0.64 25 ng/mL — 6.19 ± 0.53^(€) — 200 nM 3.75 ± 0.33 —  4.01 ± 0.45 —  4.28 ± 0.61 —  4.32 ± 0.53 — 25 ng/mL  25nM  6.15 ± 0.47 >0.999  6.00 ± 0.46 0.9967  5.65 ± 0.44 0.7981   5.4 ±0.45 0.5462 25 ng/mL  50 nM  5.57 ± 0.53 0.7712  6.22 ± 0.42 >0.999 5.41 ± 0.33 0.5151   6.1 ± 0.36 0.9997 25 ng/mL 100 nM  5.63 ± 0.390.8269  6.21 ± 0.48 >0.999  5.32 ± 0.46 0.4157  5.83 ± 0.34 0.9448 25ng/mL 200 nM  5.25 ± 0.45 0.4653  6.27 ± 0.56 0.9999  5.04 ± 0.36 0.1833 5.42 ± 0.52 0.5691 STAT5A — — 2.17 ± 0.54 25 ng/mL — 26.41 ± 2.26^(¥) —200 nM  1.12 ± 0.19 —  1.44 ± 0.41 —  1.75 ± 0.44 —  1.99 ± 0.51 — 25ng/mL  25 nM 19.04 ± 1.94 0.0111 23.69 ± 1.63 0.7471 22.82 ± 1.77 0.452020.12 ± 1.29 0.0428 25 ng/mL  50 nM 16.18 ± 1.66 0.0003 22.32 ± 2.160.4225 20.71 ± 1.77 0.1117 22.69 ± 1.71 0.3629 25 ng/mL 100 nM 12.94 ±1.27 <.0001 20.87 ± 2.1  0.1784 18.44 ± 1.85 0.0138 19.54 ± 1.34 0.023325 ng/mL 200 nM  9.48 ± 0.86 <.0001  19.2 ± 1.94 0.0505 17.64 ± 1.460.0059 18.33 ± 1.83 0.0059 STAT6 — — 749.34 ± 20.85 25 ng/mL — 1045.99 ±26.73^(¥) — 200 nM 723.56 ± 20.76 — 740.11 ± 34.98 — 762.04 ± 9.44  —777.03 ± 29.31 — 25 ng/mL  25 nM 1043.96 ± 20.37  >0.999 1004.82 ±23.76  0.5557 1020.89 ± 23.57  0.8238 1042.76 ± 29.23  >0.999 25 ng/mL 50 nM 1016.85 ± 25.68  0.8028 990.05 ± 21.06 0.2895 982.62 ± 14.340.1389 1046.46 ± 29.12  >0.999 25 ng/mL 100 nM 966.76 ± 28.58 0.0739987.64 ± 15.75 0.2557 943.66 ± 25.99 0.0059  985.1 ± 39.79 0.3955 25ng/mL 200 nM 976.22 ± 14.93 0.1487 985.17 ± 29.31 0.224 966.51 ± 12.3 0.0429 1013.25 ± 17.15  0.8453 IL-1α — — 95.72 ± 5.84 25 ng/mL — 1405.01± 27.93^(¥) — 200 nM 84.51 ± 7.04 — 85.16 ± 6.50 — 88.72 ± 5.90 — 92.67± 5.54 — 25 ng/mL  25 nM 1115.1 ± 18.96 <.0001 1288.02 ± 20     0.00471370.52 ± 35.28  0.8379 1269.66 ± 50.59  0.0744 25 ng/mL  50 nM 962.51 ±23    <.0001 1258.76 ± 23.63  0.0003 1308.7 ± 45.12 0.0995 1336.95 ±50.97  0.5871 25 ng/mL 100 nM 839.16 ± 21.04 <.0001 1162.35 ± 23.34 <.0001 1194.29 ± 12.27  <.0001 1244.96 ± 41.03  0.0264 25 ng/mL 200 nM755.65 ± 16.88 <.0001 1126.94 ± 26.22  <.0001 1151.31 ± 20.01  <.0011163.14 ± 26.71  0.0004 IL-6 — — 5.86 ± 0.38 25 ng/mL — 170.83 ±5.28^(¥) — 200 nM  4.70 ± 0.32 —  4.97 ± 0.36 —  4.98 ± 0.28 —  5.15 ±0.31 — 25 ng/mL  25 nM 93.79 ± 4.03 <.0001 130.24 ± 3.84  <.0001 135.32± 3.36  <.0001 132.28 ± 7.41  <.0001 25 ng/mL  50 nM  69.7 ± 2.81 <.0001122.69 ± 4.36  <.0001 128.14 ± 6.83  <.0001 137.61 ± 5.87  0.0006 25ng/mL 100 nM 51.01 ± 1.57 <.0001 111.07 ± 4.74  <.0001 112.13 ± 3.37 <.0001 122.46 ± 5.35  <.0001 25 ng/mL 200 nM 40.39 ± 2.19 <.0001 93.03 ±3.25 <.0001 101.17 ± 2.91  <.0001 119.49 ± 4.42  <.0001 ^(a)Stimulationwith TNFα (25 ng/mL) and IFNγ (25 ng/mL) ^(b)Data is presented as mean ±standard error ^(c)Significant differences compared back to stimulationwith TNFα and IFNγ alone ^(¥)Indicates significant difference of p <0.0001 from vehicle (no stimulation and no drug concentration) alone^(€)Indicates significant difference of p < 0.1 from vehicle

FIGS. 1-4 illustrate the individual gene expression values (MFI) forJAK1, JAK2, IL-1α, and IL-6, respectively, for each experimentalreplicate in keratinocytes simulated with TNFα and IFN-γ in thepresence/absence of JAK inhibitors.

Target proteins of interest in the media were detected and quantifiedusing the ProCarta Multiplex Immunoassay reagents and protocols(Invitrogen, Catalog # EPX450-12171-901). Media was incubated withantibody conjugated beads designed to bind to the epitopes of specifictarget proteins and identify the bound protein through the bead'sdistinctive spectral pattern. Biotinylated detection antibodies,designed to bind to different epitopes of the same target proteins, andStreptavidin-PE are added to assay plates to quantify the amount of thetarget proteins. Assay plates were read on the Luminex 200 and data wereexpressed as Net Median Fluorescence Intensity. The net medianflorescence values for the antigen standard curve, prepared according tothe manufacturer's procedures (Invitrogen, Catalog # EPX450-12171-901)was plotted against the expected concentrations for each standard. Theconcentration of each protein was extrapolated from the antigen standardcurve and concentrations were expressed as pg/mL (Table 5).

TABLE 5 Concentrations of Inflammatory Mediators Produced by HumanKeratinocyte cells Stimulated with TNFα and IFNγ in the Presence/Absenceof JAK Inhibitors Drug Compound A Compound B Compound C Compound DStimu- Concen- p- p- p- p- Protein lation^(a) tration pg/mL^(b)value^(c) pg/mL^(b) value^(c) pg/mL^(b) value^(c) pg/mL^(b) value^(c)IL-1α — — 0.29 ± 0.03 25 ng/mL — 7.82 ± 0.18^(¥) — 200 nM  0.26 ± 0.05 — 0.29 ± 0.03 —  0.30 ± 0.05 —  0.31 ± 0.04 — 25 ng/mL  25 nM  5.93 ±0.29 <.0001  7.34 ± 0.31 0.7043  7.74 ± 0.36 0.9994   6.8 ± 0.39 0.149825 ng/mL  50 nM  4.9 ± 0.3 <.0001  7.06 ± 0.37 0.3281  7.01 ± 0.390.3537 6.76 ± 0.4 0.1249 25 ng/mL 100 nM  4.12 ± 0.26 <.0001     7 ±0.41 0.2631  7.27 ± 0.47 0.6747 6.92 ± 0.4 0.2281 25 ng/mL 200 nM  3.45± 0.23 <.0001  6.16 ± 0.35 0.0034  6.45 ± 0.38 0.0358   6.3 ± 0.350.0121 IL-6 — — 30.57 ± 2.89 25 ng/mL — 862.33 ± 17.95^(¥) — 200 nM26.86 ± 2.62 — 28.49 ± 2.89 — 28.79 ± 2.91 — 28.84 ± 1.89 — 25 ng/mL  25nM  594.5 ± 25.17 <.0001 749.64 ± 32.94 0.0158 774.87 ± 31.09 0.1794743.07 ± 36.3  0.0476 25 ng/mL  50 nM 446.35 ± 19.73 <.0001 674.21 ±27.15 <.0001 710.89 ± 36.7  0.006 698.04 ± 29.79 0.0037 25 ng/mL 100 nM362.14 ± 18.73 <.0001  643.8 ± 27.14 <.0001  690.4 ± 35.25 0.0016 703.99± 42.22 0.0054 25 ng/mL 200 nM 295.21 ± 15.22 <.0001 568.73 ± 24.74<.0001 621.79 ± 33.44 <.0001  646.2 ± 32.46 <.0001 IP-10/ — — 20.14 ±0.36 CXCL10 25 ng/mL — 3935.46 ± 375.68^(¥) — 200 nM 19.75 ± 0.42 —19.83 ± 0.40 — 20.23 ± 0.48 — 20.39 ± 0.57 — 25 ng/mL  25 nM 3497.56 ±194.81 0.6232 4068.98 ± 507.12 0.9982 3999.39 ± 370.53 0.9998 3903.67 ±366.97 >0.999 25 ng/mL  50 nM 3599.04 ± 402.58 0.7995 3872.74 ± 295.010.9999  3665.2 ± 277.11 0.9431 3998.62 ± 456.34 0.9999 25 ng/mL 100 nM3158.24 ± 189.25 0.1574  4050.7 ± 471.31 0.999 3860.41 ± 323.05 0.99954100.26 ± 502.48 0.9978 25 ng/mL 200 nM 2662.18 ± 89.27  0.0059 4071.78± 411.22 0.9979 3835.78 ± 304.58 0.9984 4407.56 ± 645.63 0.8945 MIP1α —— 3.14 ± 0.24 25 ng/mL — 105.63 ± 3.74^(¥) — 200 nM  2.63 ± 0.35 —  2.75± 0.26 —  2.90 ± 0.21 —  3.11 ± 0.28 — 25 ng/mL  25 nM 82.56 ± 3.1 <.0001 103.81 ± 3.29  0.9925 101.71 ± 3.84  0.931 102.06 ± 4.18  0.930325 ng/mL  50 nM 70.57 ± 3.32 <.0001 100.64 ± 4.66  0.7866 104.54 ± 6.56 0.9994 96.35 ± 3.57 0.3335 25 ng/mL 100 nM 50.91 ± 1.6  <.0001 91.52 ±5.05 0.0532  96.4 ± 4.18 0.4229 96.22 ± 3.58 0.3215 25 ng/mL 200 nM40.36 ± 0.88 <.0001  83.1 ± 2.77 0.0007 98.72 ± 3.87 0.6469 88.49 ± 5.060.016 RANTES — — 9.56 ± 0.56 25 ng/mL — 230.17 ± 9.43^(¥) — 200 nM 10.17± 0.54 —  8.42 ± 0.51 —  8.61 ± 0.52 —  9.51 ± 0.56 — 25 ng/mL  25 nM   192 ± 12.74 0.0311 203.77 ± 12.55 0.4195 216.88 ± 13.45 0.9096 237.57± 17.46 0.9967 25 ng/mL  50 nM 165.12 ± 11.76 0.0001 198.35 ± 15.1 0.262 201.93 ± 15.44 0.439 237.55 ± 21.78 0.9967 25 ng/mL 100 nM 136.24± 7.8   <.0001 194.21 ± 12.67 0.1736 207.79 ± 17.38 0.6354 241.39 ±22.79 0.9841 25 ng/mL 200 nM 111.94 ± 6.48  <.0001 183.18 ± 13.92 0.0416189.62 ± 13.78 0.1403 238.51 ± 23.12 0.9942 ^(a)Stimulation with TNFα(25 ng/mL) and IFNγ (25 ng/mL) ^(b)Data is presented as mean ± 1standard error ^(c)Significant differences compared back to stimulationwith TNFα and IFNγ alone ^(¥)Indicates significant difference of p <0.0001 from vehicle (no stimulation and no drug concentration) alone

FIGS. 5 and 6 illustrate the individual protein concentrations (pg/mL)for IL-1α and IL-6, respectively, for each experimental replicate inkeratinocytes simulated with TNFα and IFN-γ in the presence/absence ofJAK inhibitors.

Example E: Hidradenitis Suppurativa Skin Biopsies are Characterized byIncreased Janus Kinase Expression

Healthy Control skin total RNA from 3 single donors was purchased fromAmsbio (Catalog #s HR101 and R1234218-50). Healthy Control skin totalRNA from a pool of donors was purchased from Life TechnologiesCorporation (Catalog # QS0639). Hidradenitis Suppurativa Skin Biopsies(41 donors) were purchased from Discovery Life Sciences as formalinfixed paraffin embedded (FFPE) blocks from which total RNA was purified.

Gene expression from the Healthy Control (n=4) and HidradenitisSuppurativa (n=41) skin total RNA samples was measured for genesoutlined in Table 6 using the QuantiGene Plex Assay reagents andprotocols (Life Technologies Corporation, Catalog # QGP-277-M19012402).Purified RNAs were used at the recommended assay range of 50 ng to 500ng and were incubated overnight with capture beads designed tospecifically hybridize with mRNA from selected genes (Table 6). Thispanel of targets included several housekeeping genes were used fornormalization of the results. After overnight incubation the signal wasamplified using branched DNA methodologies, according to themanufacturer's procedures (Life Technologies Corporation). The assayplate was read on a Luminex 200 and the data were expressed as NetMedian Fluorescence Intensity (net MFI). Data was normalized to thegeometric mean of the net MFI for the housekeeping genes ACTB and GAPDH.FIGS. 7-9 illustrate the gene expression of JAK1, JAK3, TYK2, STAT1,STAT2, STAT3, IRAK1, IRAK2, and IRAK4 in the skin of healthy controlsand subjects with hidradenitis suppurativa.

TABLE 6 Targeted genes Gene Identifier Gene Name JAK1 Janus kinase 1JAK2 Janus kinase 2 JAK3 Janus kinase 3 IRAK1 interleukin 1 receptorassociated kinase 1 IRAK2 interleukin 1 receptor associated kinase 2IRAK4 interleukin 1 receptor associated kinase 4 STAT1 signal transducerand activator of transcription 1 STAT3 signal transducer and activatorof transcription 3 STAT4 signal transducer and activator oftranscription 4 STAT5A signal transducer and activator of transcription5A STAT6 signal transducer and activator of transcription 6 STAT2 signaltransducer and activator of transcription 2 STAT5B signal transducer andactivator of transcription 5B TYK2 tyrosine kinase 2 SYK spleenassociated tyrosine kinase GAPDH glyceraldehyde-3-phosphatedehydrogenase ACTB actin beta

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication, including all patent, patent applications, andpublications, is incorporated herein by reference in its entirety.

What is claimed is:
 1. A method of treating hidradenitis suppurativa ina patient in need thereof, comprising administering to the patient atherapeutically effective amount of a compound which inhibits JAK1and/or JAK2, or a pharmaceutically acceptable salt thereof, wherein thecompound is: ruxolitinib; ruxolitinib, wherein one or more hydrogenatoms are replaced by deuterium atoms;{1-{1-[3-Fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[4-fluoro-2-(trifluoromethyl)phenyl]piperidine-1-carboxamide;[3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-1-(1-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)azetidin-3-yl]acetonitrile;4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide;((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile;3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile;3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile;4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile;4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile;[trans-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-3-(4-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperazin-1-yl)cyclobutyl]acetonitrile;{trans-3-(4-{[4-[(3-hydroxyazetidin-1-yl)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;{trans-3-(4-{[4-{[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;{trans-3-(4-{[4-{[(2R)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile;5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide;4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide;5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide;{1-(cis-4-{[6-(2-hydroxyethyl)-2-(trifluoromethyl)pyrimidin-4-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;{1-(cis-4-{[4-[(ethylamino)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;{1-(cis-4-{[4-(1-hydroxy-1-methylethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;{1-(cis-4-{[4-{[(3R)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;{1-(cis-4-{[4-{[(3S)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile;{trans-3-(4-{[4-({[(1S)-2-hydroxy-1-methylethyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;{trans-3-(4-{[4-({[(2R)-2-hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile{trans-3-(4-{[4-({[(2S)-2-hydrox;ypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;{trans-3-(4-{[4-(2-hydroxyethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile;or a pharmaceutically acceptable salt of any of the aforementioned. 2.The method of claim 1, wherein the compound or salt is selective forJAK1 and JAK2 over JAK3 and TYK2.
 3. The method of claim 1, wherein thecompound is ruxolitinib, or a pharmaceutically acceptable salt thereof.4. The method of claim 1, wherein the compound is ruxolininib, or apharmaceutically acceptable salt thereof, wherein one or more hydrogenatoms are replaced by deuterium atoms.
 5. The method of claim 1, whereinthe salt is ruxolitinib phosphate.
 6. The method of claim 1, wherein thecompound or salt is selective for JAK1 over JAK2, JAK3, and TYK2.
 7. Themethod of claim 1, wherein the compound is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,or a pharmaceutically acceptable salt thereof.
 8. The method of claim 1,wherein the salt is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileadipic acid salt.
 9. The method of claim 1, wherein the compound is4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide,or a pharmaceutically acceptable salt thereof.
 10. The method of claim1, wherein the salt is 4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-H,1′H-4,4′-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamidephosphoric acid salt.
 11. The method of claim 1, wherein the compound is((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile,or a pharmaceutically acceptable salt thereof.
 12. The method of claim1, wherein the compound is((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrilemonohydrate.
 13. The method of claim 1, wherein the compound or salt isadministered at a dosage of 15, 30, 60 or 90 mg on a free base basis.14. The method of claim 1, further comprising administering anadditional therapeutic agent.
 15. The method of claim 14, wherein theadditional therapeutic agent is an antibiotic, a retinoid, acorticosteroid, an anti-TNF-alpha agent, or an immunosuppressant. 16.The method of claim 15, wherein the antibiotic is clindamycin,doxycycline, minocycline, trimethoprim-sulfamethoxazole, erythromycin,metronidazole, rifampin, moxifloxacin, dapsone, or a combinationthereof.
 17. The method of claim 15, wherein the retinoid is etretinate,acitretin, or isotretinoin.
 18. The method of claim 15, wherein thecorticosteroid is triamcinolone, dexamethasone, fluocinolone, cortisone,prednisone, prednisolone or flumetholone.
 19. The method of claim 15,wherein the anti-TNF-alpha agent is infliximab, etanercept, oradalimumab.
 20. The method of claim 15, wherein the immunosuppressant ismethotrexate, cyclosporin A, mycophenolate mofetil, or mycophenolatesodium.
 21. The method of claim 14, wherein the additional therapeuticagent is finasteride, metformin, adapalene, or azelaic acid.
 22. Themethod of claim 1, wherein the administrating of the compound or salt istopical.
 23. The method of claim 1, wherein the administering of thecompound or salt is oral.
 24. The method of claim 1, wherein the methodresults in a 10%, 20%, 30%, 40%, or 50% improvement in HiSCR(Hidradenitis Suppurativa Clinical Response).